Neurotrophic factors are natural proteins, found in the nervous system or in non-nerve tissues innervated by the nervous system, whose function is to promote the survival and maintain the phenotypic differentiation of nerve and/or glial cells (Varon and Bunge 1978 Ann. Rev. Neuroscience 1:327; Thoenen and Edgar 1985 Science 229:238). Because of this physiological role, neurotrophic factors may be useful in treating the degeneration of nerve cells and loss of differentiated function that occurs in a variety of neurodegenerative diseases, such as Alzheimer's or Parkinson's diseases, or after traumatic injuries, such as stroke or physical trauma to the spinal cord (Appel 1981 Ann. Neurology 10:499).
In order for a particular neurotrophic factor to be potentially useful in treating nerve damage, the class or classes of damaged nerve cells must be responsive to the factor. Different neurotrophic factors typically affect distinctly different classes of nerve cells. Therefore, it is advisable to have on hand a variety of different neurotrophic factors to treat each of the classes of damaged neurons that may occur with different forms of disease or injury. A given neurotrophic factor, in addition to having the correct neuronal specificity, must be available in sufficient quantity to be used as a pharmaceutical treatment. Also, since neurotrophic factors are proteins, it would be desirable to administer to human patients only the human form of the protein, to avoid an immunological response to a foreign protein.
Since neurotrophic factors are typically present in vanishingly small amounts in tissues (e.g., Hofer and Barde 1988 Nature 331:261; Lin et al. 1989 Science 246:1023) and since human tissues are not readily available for extraction, it would be inconvenient to prepare pharmaceutical quantities of human neurotrophic factors directly from human tissues. As an alternative, it would be desirable to isolate the human gene for a neurotrophic factor and use that gene as the basis for establishing a recombinant expression system to produce potentially unlimited amounts of the human protein.
Two neurotrophic factors have been described that are closely related in amino acid sequence but which affect different, although partially overlapping, sets of responsive neurons (Leibrock et al. 1989 Nature 341:149). These two neurotrophic factors are: (1) nerve growth factor (NGF) and (2) brain-derived neurotrophic factor (BDNF). Both NGF and BDNF are apparently synthesized as larger precursor forms which are then processed, by proteolytic cleavages, to produce the mature neurotrophic factor (Edwards et al, 1986 Nature 319:784; Leibrock et al. 1989 ibid.). The only genes for members of the proposed NGF/BDNF family of neurotrophic proteins that have been reported to date are the human and various animal genes for NGF (Scott et al. 1983 Nature 302:538; Ullrich et al. 1983 Nature 303:821; Meier et al. 1986 EMBO J. 5:1489) and the pig gene for BDNF (Leibrock et al. 1989 ibid.). There is a significant similarity in amino acid sequences between mature NGFs and mature BDNF, including the relative position of all six cysteine amino acid residues, which is identical in mature NGFs and BDNF from all species examined (Leibrock et al 1989 ibid.). This suggests that the three-dimensional structure of these two proteins, as determined by the location of disulfide bonds, is similar. Both mature proteins also share a basic isoelectric point (pI). NGF and BDNF are neurotrophic factors for different, although partially overlapping, sets of responsive neurons.
Therefore, NGF and BDNF appear to define a family of structurally related neurotrophic proteins which are likely to differ in their physiological role in the organism, each member affecting a different set of responsive neurons. It would be desirable to isolate the genes for any and all additional members of this NGF/BDNF family, in order to have a battery of neurotrophic proteins available to treat the range of different nerve cell types whose functions are compromised in various forms of damage to the nervous system.
NGF is a neurotrophic factor for cholinergic neurons in the basal forebrain, among others (Hefti and Will 1987 J. Neural Transm. [Suppl] (AUSTRIA) 24:309). The functional inactivation and degeneration of the basal forebrain cholinergic neurons responsive to NGF in the course of Alzheimer's disease is thought to be the proximate cause of the cognitive and memory deficits associated with that disease (Hefti and Will 1987 ibid.). NGF has been shown to prevent the degeneration and restore the function of basal forebrain cholinergic neurons in animal models related to Alzheimer's disease, and on this basis has been proposed as a treatment to prevent the degeneration and restore the function of these neurons in Alzheimer's disease (Williams et al. 1986 Proc. Natl. Acad. Sci. USA 83:9231; Hefti 1986 J. Neuroscience 6:2155; Kromer 1987 Science 235:214; Fischer et al 1987 Nature 329:65).
BDNF is a neurotrophic factor for sensory neurons in the peripheral nervous system (Barde 1989 Neuron 2:1525). On this basis, it is possible that BDNF may prove useful for the treatment of the loss of sensation associated with damage to sensory nerve cells that occurs in various peripheral neuropathies (Schaumberg et al, 1983 "Disorders of Peripheral Nerves" F. A. Davis Co., Philadelphia, Pa.). NGF and BDNF may be shown in the future to have additional neurotrophic effects that indicate their potential usefulness in treating other kinds of nerve system damage. Also, new members of the NGF/BDNF family of neurotrophic proteins may support additional neuronal populations and, therefore, be of value in treating yet additional kinds of nerve damage.
In accordance with the principle expressed above that one should administer only human proteins to human patients, it would be desirable to obtain the human gene for BDNF in order to manufacture the human protein. Also in accordance with this principle and with the principle expressed above that it would be desirable to have a battery of neurotrophic proteins with differing neuronal specificities to treat a variety of neurological conditions, it would be desirable to obtain the human genes for any and all additional members of the NGF/BDNF family of neurotrophic proteins.
Recombinant expression systems that are capable of producing the large quantities of fully-biologically-active and structurally-unmodified mature NGF needed for pharmaceutical development and for the treatment of patients have not generally been described. See, however, European Patent Publication EP 89113709, describing the recombinant expression of NGF in insect cells. Mature NGF with these properties can be produced when human or animal NGF genes are expressed in eukaryotic cell expression systems (e.g., Edwards et al. 1988 Molec. Cell. Biol. 8:2456). In such systems, the full-length NGF precursor is first synthesized and then proteolytically processed to produce mature NGF which is correctly folded 3-dimensionally and is fully biologically active. However, eukaryotic cell expression systems in general, and specifically those reported for NGF, produce relatively low yields of NGF per gram of cells and are relatively expensive to use in manufacturing.
In contrast, expression systems that use prokaryotic cells, such as bacteria, generally yield relatively large amounts of expressed protein per gram of cells and are relatively inexpensive to use in manufacturing. However, an adequate bacterial expression system capable of producing fully-biologically-active and structurally-unmodified mature NGF has not been described (a bacterial expression system is disclosed in Canadian Patent No. 1,220,736). This failure can probably be traced to problems associated with bacterial expression systems in general and problems associated with the specific techniques employed to produce NGF in bacteria.
Bacteria are not able to correctly process precursor proteins, such as the precursor protein for NGF, by making appropriate proteolytic cleavages in order to produce the correct smaller mature protein. Therefore, to produce mature NGF in bacteria, it is necessary to express only that portion of the NGF DNA sequence encoding the mature protein and not that for the larger precursor form. When this was done in the bacterium Escherichia coli, relatively large amounts of the mature human NGF protein were produced (see, e.g., Iwai et al. 1986 Chem. Pharm. Bull. 34:4724; Dicou et al. 1989 J Neurosci, Res. 22:13). Unfortuntely, the bacterially-expressed protein had little or no biological activity.
The likely reason for this lack of biological activity is that the mature NGF protein was unable to assume spontaneously the correct 3-dimensional structure and form the correct intramolecular disulfide bonds, both of which are essential for biological activity. Therefore, it would appear necessary to develop a refolding protocol capable of restoring to the mature NGF produced in bacteria the 3-dimensional structure and intramolecular disulfide bonding pattern required for full biological activity.
A refolding protocol has been descried in European Patent Application 336,324 which restores some biological activity to mature NGF produced in bacteria. However, this protocol has serious deficiencies. The protocol uses exposure to high pH (pH 13 is recommended)--apparently to break disulfide bonds that may have formed incorrectly in the bacterially-produced NGF--followed by lowering of the pH--apparently to allow the opportunity for the correct intramolecular disulfide bonds to form. Exposure to high pH, as used in this protocol, is known to cause extensive modification of proteins, including the elimination of amine side chains in glutamine and asparagine (of which there are 7 in mature human NGF) and extensive chemical alteration of asparagine-glycine, asparagine-serine and asparagine-threonine adjacent pairs (of which there are 2 in mature human NGF). In addition to these chemical modifications, the refolding procedure appeared to restore only approximately one-tenth of the biological activity of NGF. The protocol described in European Patent application 336,324 would, therefore, appear to be inadequate to produce fully-biologically-active and chemically-unmodified mature human NGF. Although numerous protocols for refolding and renaturing proteins that do not involve harsh conditions exist, no such procedure has been applied successfully to NGF.
Therefore, mature human NGF has been unavailable in sufficient amounts for pharmaceutical use, due apparently to the inadequate production capacity and cost of eukaryotic expression systems and the inability of the bacterial expression systems so far described to produce biologically-active and chemically-unmodified mature NGF. Since human mature NGF is perceived as having a potential usefulness in the treatment of Alzheimer's disease, the unavailability of this material has been keenly felt by the scientific and clinical communities. The unavailability of biologically-active human mature NGF was seen by a panel of leading scientists, assembled by the National Institute on Aging, as the critical block to further development of NGF as a treatment for Alzheimer's disease (Phelps et al. 1989 Science 243:11).
It is presumed that similar manufacturing difficulties would apply to each member of the NGF/BDNF family of neurotrophic proteins, since members of this family so far described have identically located cysteine amino acid residues and presumably, therefore, form a pattern of intramolecular disulfide bonds identical to that of NGF (Angeletti et al. 1973 Biochemistry 12:100). Based on this consideration, a manufacturing system capable of producing fully-biologically-active and chemically-unmodified human mature NGF in large amounts in bacteria will be useful in producing similar large amounts of any member of the NGF/BDNF family in a biologically-active and unmodified form suitable for pharmaceutical use.
In view of the apparent value of such neurotrophic proteins and the current inability to produce biologically active proteins as indicated above, it would be desirable to provide the following: (1) methods for securing the genes for any and all additional neurotrophic proteins that are structurally related to NGF and BDNF in a manner similar to the way these two proteins are related to each other; (2) methods for obtaining the human genes for all members of the NGF/BDNF family for which this has not been done, including the human gene for BDNF; (3) methods for using the human genes to establish recombinant expression systems in microorganisms such as E. coli that will produce significant quantities of the mature (processed) form of each of these human proteins; and, (4) a procedure for refolding and renaturing the recombinant mature proteins to allow them to attain a biological specific activity, expected for members of this class of neurotrophic proteins.